The specific aims of this research proposal are to determine the biological and mechanical response to prolotherapy (a popular, injection-based alternative therapy). Incomplete healing of ligaments can head to chronic pain, joint instability, joint laxity, and eventually osteoarthritis. Limited clinical and basic science studies suggest that prolotherapy improves function of lax ligaments and tendons via an inflammatory reaction. We propose two animal model studies to assess mechanical and biological effects of prolotherapy. The first study utilizes uninjured rat medial collateral ligaments (MCLs). One of three prolotherapy agents (or saline or needle stick controls) is injected into the tibial insertion of the MCL. The second study creates a reproducible subfailure damage injury, which is treated with prolotherapy injections. Quantitative immunohistochemcial techniques are used to determine the presence of macrophages and neutrophils. Mechanical testing techniques are used to assess the change in function (laxity), failure force, stress, and strain, stiffness, and cross-sectional area. The results of this study will evaluate the beneficial effects of prolotherapy on stretch injured ligaments and provide possible mechanisms for clinically observed behaviors. [unreadable] [unreadable]